Methods and Systems for Predicting Whether a Subject Has a Cervical Intraepithelial Neoplasia (CIN) Lesion from a Suspension Sample of Cervical Cells

ABSTRACT

Methods of predicting whether a subject has a cervical intraepithelial neoplasia (CIN) lesion are provided. Aspects of the methods include obtaining both morphometric and biomarker data from a liquid cervical cellular sample and then using both types of data to predict whether the subject has a CIN lesion. Also provided are systems that find use in practicing the methods. The methods and systems find use in a variety of applications, including cervical cancer screening applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e) this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 61/484,142filed May 9, 2011 and U.S. Provisional Patent Application Ser. No.61/413,302 filed on Nov. 12, 2010; the disclosures of which applicationsare herein incorporated by reference.

INTRODUCTION

The Papanicolaou (PAP) smear has been the cornerstone of cervical cancerscreening since 1949. By definition, the PAP smear is a stain performedon cells smeared on a slide and visualized by microscopy. Following theadvent of liquid-based cervical cytology (LBC), cells from the cervixwere obtained using a brush, suspended in a fixative solution, and thenapplied to a slide prior to staining. Highly trained cytotechnologistsand cytopathologists review the stained slides looking for evidence ofabnormal cells as indicated by the characteristics in Table 1.

TABLE 1 Low grade squamous High grade squamous intraepithelialintraepithelial lesion lesion (LSIL) (HSIL) Caviation (Moderate-Markedincrease) N/C* Ratio (Mild increase) N/C Ratio Coarse ChromatinHyperchromasia Hyperchromasia Irregular Nuclear Contour Single Abn CellsSyncytial Aggregates Pleomorphism *N/C = nuclear to cytoplasmic ratio.

Because of the necessity to use slides for the PAP smear, otherbiomarkers used for cervical cancer screening, especially those used forthe molecular detection of HPV DNA, are performed on a separate aliquotof the LBC. Though some biomarkers such as p16 can be performed on aslide, the throughput is not desirable to accommodate the 60-70 millioncervical cytology specimens obtained every year in the US and the 150+million samples worldwide. Further, molecular techniques performed on aslide are cumbersome and time consuming, characteristics that are notamenable to cervical cancer screening.

Clinically, the PAP smear and HPV testing are used together though theyare very disparate technologies. The PAP smear has relatively lowsensitivity (50%) and relatively high specificity (90%) for high gradecervical lesions (pre-cervical cancer and cervical cancer). Conversely,HPV DNA testing has high sensitivity (>90%) but low specificity (30%)for high grade cervical lesions (pre-cervical cancer and cervicalcancer. These performance characteristics have supported the combineduse of these tests for effective cervical cancer screening. Someinvestigators have pushed for the sole use of HPV detection for cervicalcancer screening (primary HPV screening), however, current HPV DNA testslack the specificity afforded by morphologic assessment using PAP smear,raising a concern for an overwhelming number of unnecessarycolposcopy/biopsy procedures. Thus the replacement of the PAP smear byHPV testing alone remains extremely controversial.

SUMMARY

Methods of predicting whether a subject has a cervical intraepithelialneoplasia (CIN) lesion are provided. Aspects of the methods includeobtaining morphometric data as well as biomarker data and/ornon-specific cell data from a liquid cervical cellular sample byassaying the sample in suspension, and then using the different types ofdata to predict whether the subject has a CIN lesion. Also provided aresystems that find use in practicing the methods. The methods and systemsfind use in a variety of applications, including cervical cancerscreening applications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows instrument set-up for using Aspect Ratio versus Area dotplot to identify intact single cells (gated) in a liquid-based cervicalcytology (LBC) specimen.

FIG. 2 shows identification of abnormal cells in a LBC specimen by N/C(nuclear to cytoplasmic) ratio analysis using the instrument set up asin FIG. 1. Increased N/C ratios are indicative of abnormal cells.

FIGS. 3A and 3B show exemplary data obtained for single cells in a LBCspecimen. For each cell, a brigthfield image, an image showing E6/E7mRNA hybridization (FITC), an image showing the DNA quantification(DAPI), and an image showing a composite of the E6/E7 and DAPI imagesare shown. FIG. 3A shows that cells having a high N/C ratio are E6/E7positive and have abnormal DNA content. FIG. 3B shows that cells havinga low N/C ratio are E6/E7 negative and have a normal DNA content.

FIG. 4 is a dot plot showing that overexpression of E6/E7 mRNA is foundin cells having increased DNA content. Cell cycle analysis wasdetermined using a DNA-staining reagent (DAPI; X-axis) and E6/E7 mRNAwas detected by hybridization of E6/E7 mRNA probes (FITC; Y-axis).Abnormal cells (having E6,E7 overexpression and increased DNA content)are indicated in the gate.

FIGS. 5A and B show dot plots showing N/C ratios (Y axis) and DNAcontent (x axis) of LBC specimens to identify abnormal cells. FIG. 5Ashows dot plots of N/C ratios versus DNA content of normal cervicalcells (top panel) and LSIL cervical cells (bottom panel). FIG. 5B showsdot plots of N/C ratios versus DNA content of HSIL cervical cells (topand bottom panels). This alternative uses only 1 staining reagent, i.e.,the non-specific DNA-staining reagent.

FIG. 6 shows a dot plot of combined side (orthogonal) light scatter (Yaxis) and electronic volume (X axis) of cells in a cervical cytologysample. This plot shows that different cells in the sample can bedelineated using these morphometric parameters. The four different gatesidentify debris, ectocervical cells, endocervical cells, andpolymorphonuclear leukocytes (PMNS). Such gating can be employed to gatefor cells of interest to analyze according to aspects of the presentinvention.

DETAILED DESCRIPTION

Methods of predicting whether a subject has a cervical intraepithelialneoplasia (CIN) lesion are provided. Aspects of the methods includeobtaining morphometric as well as biomarker data and/or non-specificcell data from a liquid cervical cellular sample by assaying the samplein suspension, and then using the different types of data to predictwhether the subject has a CIN lesion. Also provided are systems thatfind use in practicing the methods. The methods and systems find use ina variety of applications, including cervical cancer screeningapplications.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace operableprocesses and/or devices/systems/kits. In addition, all sub-combinationslisted in the embodiments describing such variables are alsospecifically embraced by the present invention and are disclosed hereinjust as if each and every such sub-combination of chemical groups wasindividually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing embodiments of the invention, aspects ofembodiments of the methods will be described first in greater detail.Next, embodiments of systems that may be used in practicing methods ofthe invention are reviewed.

Methods

As summarized above, embodiments of the invention are directed tomethods of predicting whether a subject has a cervical intraepithelialneoplasia (CIN) lesion or cervical cancer. The term “CIN lesion” (alsoreferred to in the art as cervical dysplasia) is used in itsconventional sense to refer to the abnormal growth of squamous cells onthe surface of the cervix. As is known in the art, CIN lesions may behistologically graded as CIN1, CIN2/3, CIN2 and CIN3. CIN1 lesions arethose lesions that are confined to the basal 1/3 of the epithelium, andhave the least risk of developing into a cancerous lesion, relative tothe other categories of lesions. CIN2 lesions are characterized bymoderate dysplasia confined to the basal 2/3 of the epithelium. CIN3lesions (sometimes referred to by those of skill in the art as cervicalcarcinoma in situ) are categorized by the presence of severe dysplasiathat traverses more than 2/3 of the epithelium. The CIN2/3 category(i.e., CIN2+) collectively refers to both CIN2 and CIN3 lesions.

Embodiments of the invention are characterized by predicting thepresence of a CIN lesion in a subject with a high degree of sensitivityand specificity. By predicting is meant to prognosticate or foresee thepresence of a CIN lesion in the subject without actually taking a biopsyof the cervix of the subject. The terms sensitivity and specificity areused in their conventional sense. As such, sensitivity is a measure ofthe proportion of actual positives (as opposed to false positives) thatare correctly identified while specificity is a measure of negativeswhich are correctly identified. Embodiments of the invention may predictthe presence or absence of any type of CIN lesion. Embodiments of theinvention may also predict the type of CIN lesion, e.g., whether the CINlesion is a CIN1, CIN2+, CIN2 or CIN3 lesion. Embodiments of the methodsmake this prediction, e.g., whether a lesion is present, what type oflesion is present (such as whether a CIN2+ lesion is present) with ahigh degree of sensitivity and specificity. In some instances,sensitivity is 85% or more, such as 90% or more, including 95% or more.In some instances, specificity is 85% or more, such as 87% or more,including 90% or more.

Aspects of the methods include obtaining both: (1) morphometric data and(2) biomarker data and/or non-specific cell data from a labeled liquidsample of cervical cells in suspension from the subject, e.g., a liquidsample labeled with one or both of a biomarker label and a non-specificcell label. In other words, a liquid sample of cervical cells iscollected from the subject, where the cervical cells are in suspensionin a fluid medium (e.g., as described in greater detail below), isassayed to obtain both morphometric data and biomarker/non-specific celldata. Thus, a labeled liquid sample according to aspects of the methodsmay be a biomarker labeled liquid sample, a non-specific cell labeledliquid sample, or a biomarker and non-specific cell labeled liquidsample. The labeled liquid sample of cervical cells that is assayed maybe provided according to any convenient protocol.

In one embodiment, an initial fluid cervical cellular sample is preparedby taking a cellular sample from the cervix and combining it with asuitable fluid medium. Any convenient protocol for collecting cervicalcells may be employed. Examples of protocols of interest includeprotocols that employ a cervical brush or broom device to collect cellsfrom the surface of the cervix and the endocervix. Descriptions ofexamples of cervical cell collection devices that may find use inmethods of the invention are provided in U.S. Pat. Nos. 2,955,591;3,626,470; 3,815,580; 3,877,464; 3,881,464; 3,945,372; 4,127,113;4,175,008; 4,700,713; 4,754,764; 4,762,133; 4,754,764; 4,873,992;4,862,899; 4,953,560; 5,445,164; 5,787,891; 5,795,309; 6,387,058 and6,740,049.

Following collection, the cellular sample may be combined with asuitable liquid medium, as desired. Liquid mediums of interest include,but are not limited to: saline, or balanced salt, solutions (such asHanks' balanced salt solution, a minimal essential (MEM) tissue culturemedium, POLYSAL™ solution, and normal saline); cytology mediums, e.g.,Universal Collection Medium (UCM); the universal collection mediumdescribed in U.S. Pat. No. 7,371,518 (the disclosure of which is hereinincorporated by reference); Standard Transport Medium (STM), PRESERVCYT™fluid medium (Cytyc, Inc. (Boxborough, Mass.)); CytoRich™ fluid medium(TriPath, Inc. (Burlington, N.C.); and the like.

Where desired, the collected initial sample may be assessed for adequacyprior to proceeding further in the process. For example, an aliquot ofthe sample may be subjected to light scatter analysis to determinewhether adequate target cells are present in the sample, e.g., asdescribed in U.S. Pat. No. 6,329,167; the disclosure of which is hereinincorporated by reference.

Following preparation, the resultant initial fluid cervical cellularsample may be fixed and/or permeabilized as desired. As such, methods ofthe invention include fixing the cellular sample by contacting thesample with a suitable fixation reagent. Fixation reagents of interestare those that fix the cells at a desired timepoint. Any convenientfixation reagent may be employed, where suitable fixation reagentsinclude, but are not limited to: formaldehyde, paraformaldehyde,formaldehyde/acetone, methanol/acetone, IncellFP (IncellDx, Inc) etc.For example, paraformaldehyde used at a final concentration of about 1to 2% has been found to be a good cross-linking fixative. In someinstances, the cells in the sample are permeabilized by contacting thecells with a permeabilizing reagent. Permeabilizing reagents of interestare reagents that allow the labeled biomarker probes, e.g., as describedin greater detail below, to access to the intracellular environment. Anyconvenient permeabilizing reagent may be employed, where suitablereagents include, but are not limited to: mild detergents, such asTriton X-100, NP-40, saponin, etc.; methanol, and the like. It may alsobe desirable to label cells with a positive heavy metal control, e.g. aDNA intercalator labeled with a heavy metal, e.g. iridium, etc. Cellsmay also be stained with a viability dye prior to fixation, e.g.ethidium bromide, propidium iodide, DAPI, RhCl₃, etc., as desired.

In certain embodiments, and as reviewed above, the sample that isassayed to obtain morphometric and biomarker data is a biomarker labeledsampled. Accordingly, the sample is one that has been labeled for one ormore biomarkers of interest. By “biomarker labeled sample” is meant asample which has been contacted with a labeled biomarker probe (e.g., asdescribed in greater detail below) that specifically binds to abiomarker of interest if the biomarker is present in the cellularsample. Biomarkers of interest include, but are not limited to, cervicalcancer biomarkers. Cervical cancer biomarkers are a distinctivebiological or biologically derived indicator, for example nucleic acidsor proteins, whose presence is associated or linked with either thepropensity of a subject to develop cervical cancer or the presence ofcervical cancer in a subject. Accordingly, biomarkers of interestinclude nucleic acid and protein analytes whose presence and/or amountin a cell can be used to make a prediction of at least the propensity ofa subject to suffer from cervical cancer. Biomarkers of interestinclude, but are not limited to: HPV expression products of HPV genes,such as HPV genes L1, L2, E2, E4, E5, E6 or E7; cyclin-dependent kinaseinhibitors, e.g., p14, p15^(INK4b), p16 (i.e., p16^(INK4a) as describedin Serrano, M., et al., Nature, 1993 Dec. 16; 366(6456): 704-7),p18^(INK4c), p19^(INK4d), p21^(WAF1/CIP1) and p27^(KIP1); cell cycleregulatory proteins, e.g., p14^(ARF); specific microRNAs or chromosomealterations 3q-associated with cervical cancer, such as described inUnited States Patent Publication No. 20100234445 (the disclosure ofwhich is herein incorporated by reference); etc.

The biomarker labeled liquid cervical cellular sample that is assayed inmethods of the invention may be prepared using any convenient labelingprotocol. In some embodiments, preparation of the biomarker labeledliquid sample includes contacting an initial cervical cell sample with alabeled biomarker probe that specifically binds to a cervical cancerbiomarker. Depending on the particular assay to be performed, theinitial sample may be combined with a single labeled biomarker probe ortwo or more distinct labeled biomarker probes that bind to differentbiomarkers of different molecular composition, where the number of suchdistinct labeled biomarker probes may be two or more, e.g., three ormore, four or more, five or more, etc; e.g., where the assay is amultiplex assay for two or more biomarkers.

In contacting the initial sample with the labeled biomarker probe(s),the sample is combined with one or more labeled biomarker probes toproduce a reaction mixture. Labeled biomarker probes of interest includea specific binding domain and a label domain. The specific bindingdomain comprises a capture ligand that specifically binds to thebiomarker of interest. Depending on the particular assay, the biomarkerof interest may be a variety of different types of molecules, includingbut not limited to: proteins, polypeptides, proteoglycans, glycoproteinsand the respective fragments of these molecules; nucleic acids, e.g.,DNA and RNA, such as mRNA, etc. The capture ligand is therefore a ligandthat binds to the biomarker molecule of interest, wherein this captureligand may of course vary depending on the specific type of biomarkermolecule to be detected, e.g., antibody for protein biomarker,oligonucleotide for mRNA biomarker. In certain embodiments, the affinitybetween a capture ligand and the biomarker molecule to which itspecifically binds when they are specifically bound to each other in abinding complex is characterized by a K_(D) (dissociation constant) of10⁻⁶ M or less, 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁹ Mor less, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹³ M or less, 10⁻¹⁴ M orless, including 10⁻¹⁵ M or less.

As indicated above, a variety of different types of specific bindingagents may be employed as the capture ligands, where the particular typeof binding agent is selected based, at least in part, on the particulartype of molecule of the biomarker of interest. Specific binding agentsof interest include antibody binding agents, proteins, peptides,haptens, nucleic acids, etc. The term “antibody binding agent” as usedherein includes polyclonal or monoclonal antibodies or fragments thatare sufficient to bind to an analyte of interest. The antibody fragmentscan be, for example, monomeric Fab fragments, monomeric Fab′ fragments,or dimeric F(ab)′₂ fragments. Also within the scope of the term“antibody binding agent” are molecules produced by antibody engineering,such as single-chain antibody molecules (scFv) or humanized or chimericantibodies produced from monoclonal antibodies by replacement of theconstant regions of the heavy and light chains to produce chimericantibodies or replacement of both the constant regions and the frameworkportions of the variable regions to produce humanized antibodies.Nucleic acid binding agents of interest are nucleic acids thatspecifically bind to biomarker nucleic acids in a cell. The length ofthese nucleic acids may vary, so long as it is sufficient for theoligonucleotide to serve as a specific binding agent, and in someinstances ranges from 13 to 100 nt, such as 14 to 50 nt, e.g., 15 to 25nt. The oligonucleotides that make up these nucleic acid binding agentsmay be DNA or RNA, or a synthetic analogue thereof, as desired.

In addition to the specific binding domain, the labeled biomarker probesfurther include a detectable label. Of interest as detectable labels arefluorescent dyes. Fluorescent dyes (fluorophores) can be selected fromany of the many dyes suitable for use in imaging applications (e.g.,flow cytometry). A large number of dyes are commercially available froma variety of sources, such as, for example, Molecular Probes (Eugene,Oreg.) and Exciton (Dayton, Ohio). Examples of fluorophores of interestinclude, but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives such as acridine, acridine orange, acrindine yellow,acridine red, and acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BrilliantYellow; coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine andderivatives such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylaminocoumarin; diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluoresceinisothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein,and QFITC (XRITC); fluorescamine; IR144; IR1446; Green FluorescentProtein (GFP); Reef Coral Fluorescent Protein (RCFP); Lissamine™;Lissamine rhodamine, Lucifer yellow; Malachite Green isothiocyanate;4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine;pararosaniline; Nile Red; Oregon Green; Phenol Red; B-phycoerythrin;o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrenebutyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G),4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride,rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolicacid and terbium chelate derivatives; xanthene; or combinations thereof.Other fluorophores or combinations thereof known to those skilled in theart may also be used, for example those available from Molecular Probes(Eugene, Oreg.) and Exciton (Dayton, Ohio).

Where multiple distinct labeled biomarker probes are employed, the labelof each distinct probe may be chosen to provide a distinguishablesignal. For example, in embodiments where first and second distinctlabeled biomarker probes are employed, the label in the second probe isa fluorescent label which produces a fluorescent signal that isdistinguishable from the first fluorescent signal of the label of thefirst probe. Accordingly, the first and second fluorescent signalsproduced upon excitation of the first and second fluorescent labels aredistinguishable from each other, meaning that both can be detected atthe same time and that the signal from one does not modify or change thesignal from the other. Each distinct label may produce signals that aredistinguishable from any other label. For example, the cells may bestained with a third fluorescent label which produces a thirdfluorescent signal that is distinguishable from the first and secondfluorescent signals.

In some instances, the labeled biomarker probe that is employed is anHPV E6, E7 oligonucleotide fluorescently labeled probe. Such probes mayvary in length, ranging in some instances from 13 to 100 nt, such as 14to 50 nt, e.g., 15 to 25 nt. The particular sequence of such probes mayvary. Specific sequences of interest include, but are not limited tothose found in the probe cocktail of the HPV OncoTect™ E6,E7 mRNADetection Kit (incellDx, Menlo Park, Calif.).

Exemplary probe sequences specific for HPV E6/E7 gene (or mRNA) includethose described in Faulkner-Jones et al. (J. Virol Methods 1993 vol. 41pages 277-296; incorporated herein by reference). The sequences fromTable 1 of Faulkner-Jones et al., which shows such exemplary HPV E6/E7specific probe sequences, are provided below.

HPV Type Region Sequence (5′-3′) 6b/11 E6 + E7TTAGGGTAACATGTCTTCCATGCATGTTGT (SEQ ID NO: 01) E7ACGTTGCTGTCACATCCACAGCAACAGGTCA (SEQ ID NO: 02) 16 E6TGCAACAAGACATACATCGACCGGTCCACCGAC (SEQ ID NO: 03) E7GGTTACAATATTGTAATGGGCTCTCTCCGG (SEQ ID NO: 04) E6TTTCAGGACCCACAGGAGCGACCCAGAAAG (SEQ ID NO: 05) 18 E6 + E7TCGAGCACGAATGGCACTGGCCTCTATAGTGCCCAG (SEQ ID NO: 06) E7GGTCAACCGGAATTTCATTTTGGGGCTCTAAATG (SEQ ID NO: 07) 33 E6CTTGGCACAAATCATGCAATGTTCGTGGTT (SEQ ID NO: 08)

Additional exemplary probe sequences for HPV E6/E7 include thosedescribed in Plummer et al. (Diagnostic Mol. Path. 1998 vol. 7, pages76-84; incorporated herein by reference). The sequences from Table 1 inPlummer et al., which shows such exemplary HPV E6/E7 probe sequences,are provided below.

HPV Type Region Sequence (5′-3′) HPV16 E6 GTCCTGAAACATTGCAGTTCTCTTTTGGTG(SEQ ID NO: 09) E6 CTGTGCATAACTGTGGTAACTTTCTGGGTC (SEQ ID NO: 10) E6TCACACAACGGTTTGTTGTATTGCTGTTCT (SEQ ID NO: 11) E6/E7TGGGTTTCTCTACGTGTTCTTGATGATCTG (SEQ ID NO: 12) E7TAACAGGTCTTCCAAAGTACGAATGTCTAC (SEQ ID NO: 13) E7TATGGTTTCTGAGAACAGATGGGGCACACA (SEQ ID NO: 14) HPV18 E6AGTGTTCAGTTCCGTGCACAGATCAGGTAG (SEQ ID NO: 15) E6CCTCTGTAAGTTCCAATACTGTCTTGCAAT (SEQ ID NO: 16) E6CCTCTATAGTGCCCAGCTATGTTGTGAAAT (SEQ ID NO: 17) E6/E7TTGTGTTTCTCTGCGTCGTTGGAGTCGTTC (SEQ ID NO: 18) E7CTGGCTTCACACI7ACAACACATACACAAC (SEQ ID NO: 19) E7TGCTCGAAGGTCGTCTGCTGAGCTTTCTAC (SEQ ID NO: 20)

Additional HPV specific probe sequences that find use in the methods andsystems described herein may be designed and tested using any convenientprobe design methodology.

In preparing the reaction mixture, the sample may be combined with thelabeled biomarker probes using any convenient protocol. Combination maybe carried out with mixing, as desired. Contact of the sample with thelabeled biomarker probes is performed under incubation conditions thatprovide for binding of probes to their respective biomarkers, ifpresent, in the sample. In some instances, the probes and samples arecontacted and combined at a temperature ranging from 15 to 50, such asfrom 20 to about 40° C. Contact may be performed with mixing oragitation, e.g., with vortexing etc., to provide for sufficientcombination of the reaction components and the sample.

The resultant reaction mixture may then be maintained or incubated for aperiod of time prior to assay for morphometric and biomarker data, e.g.,via flow cytometric analysis (e.g., as described in greater detailbelow). In some instances, the reaction mixture is incubated at atemperature ranging from 15 to 50, such as from 20 to about 40° C. for aperiod of time ranging from about 30 minutes to 72 hours, such as 1 hourto 24 hours, including 1 hour to 3 hours. Following the above incubationstep, the sample may be assayed immediately or stored for assay at alater time. If stored, in some embodiments the sample is stored at areduced temperature; e.g., on ice.

Where desired, the resultant reaction mixture may be washed, e.g., toremove any unbound probes and other sample components. Washing may beperformed using any convenient protocol, e.g., by combining the reactionmixture with a suitable wash buffer and separating the cells from thefluid. A given washing protocol may include one or more distinct washingsteps, as desired. Following any washing protocol, the labeled cells maybe re-suspended in a suitable liquid, e.g., the washing buffer oranother buffer, for subsequent analysis, e.g., via flow cytometricanalysis.

In some embodiments, as described above, the labeled liquid cell sampleis labeled (or stained) with a non-specific cell stain, either inaddition to or in the absence of a biomarker label. The cells may bestained with a non-specific stain using any convenient protocol. Ofinterest as non-specific cells stains are DNA specific stains. Dyes andstains that are specific for DNA (or preferentially bind double strandedpolynucleotides in contrast to single-stranded polynucleotides) andtherefore may be employed as non-specific stains include, but are notlimited to: Hoechst 33342(2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole)and Hoechst 33258(2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole)and others of the Hoechst series; SYTO 40, SYTO 11, 12, 13, 14, 15, 16,20, 21, 22, 23, 24, 25 (green); SYTO 17, 59 (red), DAPI, DRAQ5™ (ananthraquinone dye with high affinity for double stranded DNA), YOYO-1,propidium iodide, YO-PRO-3, TO-PRO-3, YOYO-3 and TOTO-3, SYTOX Green,SYTOX, methyl green, acridine homodimer, 7-aminoactinomycin D,9-amino-6-chloro-2-methoxyactridine. Depending on the particular stainand assay, the stain may serve in quantitation of biomarker, as anindication of cell cycle, etc.

Following preparation of the labeled sample, e.g., as described above,the sample is assayed to obtain both morphometric as well as biomarkerand/or non-specific cell data. In some instances, the same aliquot ofsample, i.e., the same physical quantity of sample, is assayed to obtainboth the morphometric and biomarker/non-specific cell data. Accordingly,these embodiments are distinguished from protocols in which a firstaliquot of a sample is assayed using one protocol, e.g., slide basedprotocol, for morphometric data and a second aliquot of the sample isassayed using another protocol, e.g., flow cytometric protocol.

Morphometric data refers to any type of data from which cell morphologyinformation, i.e., information about the size, shape and/or structure ofthe cells, may be derived. Morphometric data of interest includes, butis not limited to data selected from the group consisting of: forwardlight scatter data, side light scatter data, image data and combinationsthereof. Morphological parameters of interest include, but are notlimited to: nuclear area, perimeter, texture or spatial frequencycontent, centroid position, shape (i.e., round, elliptical,barbell-shaped, etc.), volume, and ratios of any of these parameters.The obtained morphometric date may be for cells as a whole or forsubparts thereof, e.g., the cytoplasm of cells. In some instances, themorphometric data may include an actual designation of whether a cell isnormal or abnormal, including the type of abnormal cell. For example,morphometric data may, in some instances, include a designation that agiven cell is abnormal, for example that the cell is: an atypicalsquamous cell, e.g., atypical squamous cell of undetermined significance(ASC-US), atypical squamous cell—cannot exclude HSIL (ASC-H); low gradesquamous intraepithelial lesion (LGSIL or LSIL); high grade squamousintraepithelial lesion (HGSIL or HSIL); squamous cell carcinoma;atypical glandular cell not otherwise specified (AGC-NOS); atypicalclandular cell, suspicious for AIS or cancer (AGC-neoplastic); andadenocarcinoma in situ (AIS).

Biomarker data refers to any type of data from which biomarkerinformation for the cell may be derived. In some instances, biomarkerdata is data that includes a signal emitted by the label of the labeledbiomarker probe that is employed in the assay. The biomarker data may bein the form of the presence and amplitude of emitted light, the numberof discrete positions in a cell or other object from which the lightsignal(s) originate(s), the relative placement of the signal sources,and the color (wavelength or waveband) of the light emitted at eachposition in the cell. The biomarker data may take the form ofqualitative, semi-quantitative or quantitative data. Qualitative data issimply the presence or absence of the biomarker. Semi-quantitative orquantitative data is data that provides some indication of the amount,e.g., copy number, concentration, etc., of the biomarker in the cell.For example, semi-quantitative data may take the form of an indicationthat the copy number of a biomarker of interest is above a certainthreshold number. Quantitative data provides an indication of anabsolute value, e.g., copy number, amount, etc., of the biomarker in thecell. Semi-quantitative and quantitative data may be collectivelyreferred to as biomarker quantitation data.

In some instances, the biomarker of interest is an mRNA that is presentwhen a subject is infected with a high risk HPV strain. mRNAs ofinterest include an HPV E6, E7 mRNA. In these embodiments, the absolutecopy number of the mRNA species of interest may be determined, such thatthe quantitation data of the mRNA species of interest is obtained. Insome instances where the biomarker is HPV E6, E7, the total amount ofE6, E7 mRNA species per cell is determined. In these instances, ofinterest is the identification of cells that 2 or more, such as 5 ormore, including 10 or more, 50 or more, 100 or more, 200 or more, 500 ormore HPV E6, E7 mRNA copies per cell, as these cells may be associatedwith the presence of a CIN lesion in the host. In some instances, thebiomarker data that is obtained in methods of the invention providesinformation that there are 2 to 1000 copies of HPV E6, E7 mRNA per cell,e.g., 5 to 750 copies of HPV E6, E7 mRNA per cell, including 10 to 500copies of HPV E6, E7 mRNA per cell. In some instances, the biomarkerdata that is obtained is the HPV E6, E7 mRNA copy number per cell dataas described in U.S. Pat. No. 7,524,631, the disclosure of which isherein incorporated by reference. Determination of the copy number mayinclude comparing a single to a suitable control, e.g. as provided by anon-specific DNA stain (such as described below), by a reference value,etc.

In some instances, photometric measurements are also obtained.Photometric measurements enable the determination of nuclear opticaldensity, cytoplasm optical density, background optical density, and theratios of any of these values.

Non-specific cell data is any type of data from which non-specific(i.e., non-biomarker specific) cell information for the cells in thesample may be derived. In some instances, non-specific cell data is datathat includes a signal emitted by a non-specific cell stain that isemployed in the assay. The non-specific cell data may be in the form ofthe presence and amplitude of emitted light, the number of discretepositions in a cell or other object from which the light signal(s)originate(s), the relative placement of the signal sources, and thecolor (wavelength or waveband) of the light emitted at each position inthe cell. The non-specific cell data may take the form of qualitative,semi-quantitative or quantitative data. For example, quantitative orsemi-quantitative non-specific cell data may include data on the DNAcontent of a cell obtained using a DNA stain (e.g., as described above).Other quantitative data includes electronic volume (EV). Thus,non-specific cell data may provide an indication of an absolute orrelative value, e.g., copy number, amount, etc., of the non-specificcell component in the cell. Semi-quantitative and quantitative data maybe collectively referred to as non-specific cell quantitation data.Qualitative non-specific cell data can provide information with respectto the presence of absence of a particular feature of a cell. Forexample, in cells stained with a DNA stain, the presence or absence of acell nucleus can be determined (e.g., the number of nucleated cells in asample, also referred to as percent nucleation).

It is noted that where multiple distinct labels are to be detected in asingle labeled liquid cell sample, e.g., biomarker probe labels andnon-specific cell stains used in a single sample, the labels and stainsemployed may be chosen to provide a distinguishable signal (as describedabove for using multiple biomarker probes in a single sample, above).For example, in embodiments where a labeled biomarker probe and a DNAstain are employed, the label for the biomarker probe is a fluorescentlabel which produces a fluorescent signal that is distinguishable fromthe fluorescent signal of the DNA stain. Accordingly, the fluorescentsignals produced upon excitation of the biomarker probe label and theDNA stain are distinguishable from each other, meaning that both can bedetected at the same time and that the signal from one does not modifyor change the signal from the other. Each distinct label may producesignals that are distinguishable from any other label. For example, thecells may be stained with three fluorescent labels which produce threedistinct fluorescent signals that are distinguishable from each otherupon excitation.

The above types of data, e.g., the morphometric, biomarker, andnon-specific cell data, may be obtained from the same aliquot of sampleusing any convenient protocol. In some instances, a flow cytometricprotocol is employed which collects each type of data. Flow cytometry isa well-known methodology using multi-parameter data for identifying anddistinguishing between different particle (e.g., cell) types i.e.,particles that vary from one another terms of label (wavelength,intensity), size, etc., in a fluid medium. In flow cytometricallyanalyzing the sample prepared as described above, an aliquot of thesample is first introduced into the flow path of the flow cytometer.When in the flow path, the cells in the sample are passed substantiallyone at a time through one or more sensing regions, where each of thecells is exposed separately individually to a source of light at asingle wavelength (or in some instances two or more distinct sources oflight) and measurements of morphometric parameters, e.g., light scatterparameters, and/or biomarker parameters, e.g., fluorescent emissions, asdesired, are separately recorded for each cell. The data recorded foreach cell is analyzed in real time or stored in a data storage andanalysis means, such as a computer, for later analysis, as desired.

More specifically, in a flow cytometer, the cells are passed, insuspension, substantially one at a time in a flow path through one ormore sensing regions where in each region each cell is illuminated by anenergy source. The energy source may include an illuminator that emitslight of a single wavelength, such as that provided by a laser (e.g.,He/Ne or argon) or a mercury arc lamp with appropriate filters. Forexample, light at 488 nm may be used as a wavelength of emission in aflow cytometer having a single sensing region. For flow cytometers thatemit light at two distinct wavelengths, additional wavelengths ofemission light may be employed, where specific wavelengths of interestinclude, but are not limited to: 535 nm, 635 nm, and the like.

In series with a sensing region, detectors, e.g., light collectors, suchas photomultiplier tubes (or “PMT”), image collectors (e.g., in the formof charge-coupled devices (CCDs)), etc., are used to record light thatpasses through each cell (generally referred to as forward lightscatter), light that is reflected orthogonal to the direction of theflow of the cells through the sensing region (generally referred to asorthogonal or side light scatter) and fluorescent light emitted from thecells, if it is labeled with fluorescent marker(s), as the cells passesthrough the sensing region and is illuminated by the energy source. Eachtype of data that is obtained, e.g., forward light scatter (or FSC),orthogonal light scatter (SSC), and fluorescence emissions (FL1, FL2,etc.) and image, etc., comprise a separate parameter for each cell (oreach “event”).

Flow cytometers further include data acquisition, analysis and recordingmeans, such as a computer, wherein multiple data channels record datafrom each detector for the morphometric and biomarker data emitted byeach cell as it passes through the sensing region. The purpose of theanalysis system is to classify and count cells wherein each cellpresents itself as a set of digitized parameter values. In flowcytometrically assaying cells in methods of the invention, the flowcytometer may be set to trigger on a selected parameter in order todistinguish the cells of interest from background and noise. “Trigger”refers to a preset threshold for detection of a parameter. It istypically used as a means for detecting passage of cell through thelaser beam. Detection of an event which exceeds the threshold for theselected parameter triggers acquisition of light scatter andfluorescence data for the cell. Data is not acquired for cells or othercomponents in the medium being assayed which cause a response below thethreshold. The trigger parameter may be the detection of forwardscattered light caused by passage of a cell through the light beam. Theflow cytometer then detects and collects the light scatter andfluorescence data for cell.

A particular subpopulation of interest is then further analyzed by“gating” based on the data collected for the entire population. Toselect an appropriate gate, the data is plotted so as to obtain the bestseparation of subpopulations possible. This procedure is typically doneby plotting forward light scatter (FSC) vs. side (i.e., orthogonal)light scatter (SSC) on a two dimensional dot plot. The flow cytometeroperator then selects the desired subpopulation of cells (i.e., thosecells within the gate) and excludes cells which are not within the gate.Where desired, the operator may select the gate by drawing a line aroundthe desired subpopulation using a cursor on a computer screen. Onlythose cells within the gate are then further analyzed by plotting theother parameters for these cells, such as fluorescence.

Any flow cytometer that is capable of obtaining both the morphometricand biomarker/non-specific cell data, e.g., as described above, from thesame aliquot of a liquid sample may be employed. Of interest are thoseflow cytometer systems described in U.S. Pat. Nos. 6,211,955, 6,249,341,6,256,096, 6,473,176, 6,507,391, 6,532,061, 6,563,583, 6,580,504,6,583,865, 6,608,680, 6,608,682, 6,618,140, 6,671,044, 6,707,551,6,763,149, 6,778,263, 6,875,973, 6,906,792, 6,934,408, 6,947,128,6,947,136, 6,975,400, 7,006,710, 7,009,651, 7,057,732, 7,079,708,7,087,877, 7,190,832, 7,221,457, 7,286,719, 7,315,357, 7,450,229,7,522,758, 7,567,695, 7,610,942, 7,634,125, 7,634,126, 7,719,598; thedisclosures of which are herein incorporated by reference.

Morphometric and biomarker/non-specific cell data obtained from the samealiquot of cervical cellular sample, e.g., as described above, isemployed to whether the subject has a CIN lesion, as described above.Various combinations of morphometric, biomarker, and non-specific celldata may be employed in making a prediction of whether a subject has aCIN lesion. Combinations of interest include, but are not limited to,the following: a) nuclear to cytoplasmic (N/C) ratio analysis (e.g., toidentify abnormal cells) coupled with quantification of E6, E7 mRNA; b)N/C ratio analysis coupled with cell cycle analysis as determined byDAPI staining and green fluorescence of the E6, E7 mRNA hybridizationsignal; c) N/C ratio analysis coupled with cell cycle analysis asdetermined by p16 staining and green fluorescence of the E6, E7 mRNAhybridization signal; d) N/C ratio analysis coupled with cell cycleanalysis as determined by DAPI (or other DNA stain) staining, etc.

Increased, or high, N/C ratios include N/C ratios that are 0.25 orhigher, 0.30 or higher, 0.4 or higher, 0.5 or higher, 0.6 or higher, 0.7or higher, 0.8 or higher, 0.9 or higher, 0.95 or higher, etc.

In addition to N/C ratio, nuclear area (NA) assessment may be used asmorphometric data. Cells in a cervical cell sample having an increasednuclear area, e.g., as compared to nuclei in normal intermediatesquamous cells, can be identified cells as abnormal cells. Abnormalcells may have a nuclear area-to-nuclear area ratio (nuclear area ofcell of interest/nuclear area of normal intermediate squamous cell) of1.25 or more, 1.75 or more, 2.0 or more, 2.25 or more, 2.75 or more, 3.0or more, 3.25 or more, 3.5 or more, etc. It is noted here that usingstandard microscopy observations, the accuracy of estimating nucleararea is low (see, e.g., Schmidt et al., 2008, “Visual estimates ofnucleus-to-nucleus ratios: can we trust our eyes to use the BethesdaASCUS and LSIL size criteria?” Cancer 114(5):287-93. Aspects of thepresent invention provide a more accurate and reproducible assessment ofnuclear area.

Additional data parameters may also be employed in analyzing the cellsin the cervical cell sample, including side (orthogonal) light scatter,forward scatter, and electronic volume (EV; a measure of cell size;based on the Coulter principle). Such parameters find use in identifyingpopulations or sub-populations of cells in the sample that are to beanalyzed for the presence of abnormal cells (see, e.g., FIG. 6 and itsdescription herein). For example, analysis of EV and DNA content (e.g.,using DAPI) allows the differentiation of enucleated from nucleatedsquamous cells.

In some instances, the methods include determining that the assayedcellular sample includes cancerous cells. In these embodiments, themethods include identifying the presence of one or more cancerous cellsin the sample, where the identification is made based on morphometricand biomarker data, e.g., as described above. Such embodiments may ormay not include predicting the presence of CIN in a subject, since themethods of these embodiments identify the presence of actual cancerouscells in the sample.

In some instances, the prediction of the presence of a CIN lesion ismade within a short period of time following introduction of the sampleinto the cytometer. Accordingly, results may be provided to a user in aperiod of 6 hours or less, such as 3 hours or less, e.g., 2 hours orless, including 1 hour or less. Where desired, the overall assay timewhich ranges from obtainment of the sample from the subject to deliveryof the result to the subject is 6 hours or less, such as 5 hours orless, e.g., 4 hours less, including 3 hours or less, e.g., 2 hours orless.

In some instances, the methods further include performing furtheranalysis of a subject if the methods result in a prediction of a CINlesion in the subject. For example, where methods of the inventionresult in prediction of a CIN2+ lesion in a subject, the methods maythen further include providing a recommendation to a subject thatfurther action be taken, e.g., in the form of further diagnosticprocedures, such as biopsy. In some instances, the methods includetaking further diagnostic action. Further diagnostic action may includea colposcopy, in which a magnified visual inspection of the cervix isperformed to identify abnormal cells on the surface of the cervix. Ifthe biopsy indicates that cancer or pre-cancerous lesions may bepresent, further diagnostic and treatment procedures may be taken, suchas loop electrical excision procedure (LEEP) and conization, in whichthe inner lining of the cervix is removed to be examined pathologically.

While the methods are suitable for use with a variety of differentfemale mammalian subjects, of interest are use of the methods with humanfemale subjects, such as human female subjects of 10 years age or older,e.g., 15 years age or older, including 20 years age or older.

Devices and Systems

Aspects of the invention further include systems for use in practicingthe subject methods. Systems of interest include a flow cytometerconfigured to assay a liquid sample for both morphometric and biomarkerdata, e.g., as described above. Flow cytometers of interest include, butare not limited, to those devices described in U.S. Pat. Nos. 4,704,891;4,727,029; 4,745,285; 4,867,908; 5,342,790; 5,620,842; 5,627,037;5,701,012; 5,895,922; and 6,287,791 which, if necessary, are modified toinclude the ability to obtain image data as described above, as well asthose cytometers described in U.S. Pat. Nos. 6,211,955, 6,249,341,6,256,096, 6,473,176, 6,507,391, 6,532,061, 6,563,583, 6,580,504,6,583,865, 6,608,680, 6,608,682, 6,618,140, 6,671,044, 6,707,551,6,763,149, 6,778,263, 6,875,973, 6,906,792, 6,934,408, 6,947,128,6,947,136, 6,975,400, 7,006,710, 7,009,651, 7,057,732, 7,079,708,7,087,877, 7,190,832, 7,221,457, 7,286,719, 7,315,357, 7,450,229,7,522,758, 7,567,695, 7,610,942, 7,634,125, 7,634,126, 7,719,598; thedisclosures of which are herein incorporated by reference.

In some instances, the flow cytometer includes: a flow channel; at leasta first light source configured to direct light to an assay region ofthe flow channel (where in some instances the cytometer includes two ormore light sources); a first detector configured to receive light of afirst wavelength from the assay region of the flow channel; and a seconddetector configured to receive light of a second wavelength from theassay region of the flow channel; and an image detector configured toobtain image data of cells. Such a cytometer would have at least twodetection channels in addition to the image detector. In some instances,the device may include more than two detection channels, e.g., 3 ormore, 4 or more, 5 or more, 10 or more, etc.

Aspects of the invention further include signal processing moduleconfigured to receive the morphometric and biomarker data from the firstand second detectors and output a result of a prediction of whether asubject has a cervical intraepithelial neoplasia (CIN) lesion based onboth the morphometric data and biomarker data. The signal processingmodule may be integrated into the cytometer as a single device, ordistributed from the cytometer where the signal processing module andcytometer are in communication with each other, e.g., via a wired orwireless communication protocol.

Accordingly, aspects of the invention further include systems, e.g.,computer based systems, which are configured to predict the presence ofa CIN lesion in a subject, e.g., as described above. A “computer-basedsystem” refers to the hardware means, software means, and data storagemeans used to analyze the information of the present invention. Theminimum hardware of the computer-based systems of the present inventioncomprises a central processing unit (CPU), input means, output means,and data storage means. A skilled artisan can readily appreciate thatany one of the currently available computer-based system are suitablefor use in the present invention. The data storage means may compriseany manufacture comprising a recording of the present information asdescribed above, or a memory access means that can access such amanufacture.

To “record” data, programming or other information on a computerreadable medium refers to a process for storing information, using anysuch methods as known in the art. Any convenient data storage structuremay be chosen, based on the means used to access the stored information.A variety of data processor programs and formats can be used forstorage, e.g. word processing text file, database format, etc.

A “processor” references any hardware and/or software combination thatwill perform the functions required of it. For example, any processorherein may be a programmable digital microprocessor such as available inthe form of a electronic controller, mainframe, server or personalcomputer (desktop or portable). Where the processor is programmable,suitable programming can be communicated from a remote location to theprocessor, or previously saved in a computer program product (such as aportable or fixed computer readable storage medium, whether magnetic,optical or solid state device based). For example, a magnetic medium oroptical disk may carry the programming, and can be read by a suitablereader communicating with each processor at its corresponding station.

Embodiments of the subject systems include the following components: (a)a communications module for facilitating information transfer betweenthe system and one or more users, e.g., via a user computer, asdescribed below; and (b) a processing module for performing one or moretasks involved in the quantitative analysis methods of the invention.

In certain embodiments, a computer program product is describedcomprising a computer usable medium having control logic (computersoftware program, including program code) stored therein. The controllogic, when executed by the processor of the computer, causes theprocessor to perform functions described herein. In other embodiments,some functions are implemented primarily in hardware using, for example,a hardware state machine. Implementation of the hardware state machineso as to perform the functions described herein may be accomplishedusing any convenient method and techniques.

In addition to the sensor device and signal processing module, e.g., asdescribed above, systems of the invention may include a number ofadditional components, such as data output devices, e.g., monitorsand/or speakers, data input devices, e.g., interface ports, keyboards,etc., fluid handling components, power sources, etc.

In some instances, the systems may further include a reaction mixturederivative thereof (e.g., washed cells produced therefrom), where thereaction mixture is prepared as described above, e.g., by combining asample, one or more labeled biomarker probes and, optional, anon-specific stain.

Utility

The subject methods and systems find use in a variety of differentapplications where detection prediction of a CIN lesion in a subject isdesired. Such applications include both research and diagnosticapplications, e.g., applications where a subject is diagnosed withrespect to the presence of or propensity to develop cervical cancer,e.g., as described above. The clinical utility of the methods andsystems described herein provide powerful tools to detect and screen HPVrelated pathogenesis and cervical cancer development in both early andlate stages, thus allowing therapeutic intervention to prevent diseaseprogression as well as a chance to provide early treatment. In addition,the subject methods and systems finds use in the prognosis or riskassessment of an HPV-related disease or condition, for monitoring of theevolution of an HPV-related disease or condition, and for monitoring theefficiency of an anti-HPV drug or treatment, such as e.g., an anti-HPVvaccine or an anti-HPV vaccine candidate.

Computer Related Embodiments

Aspects of the invention further include a variety of computer-relatedembodiments. Specifically, the data analysis methods described in theprevious sections may be performed using a computer. Accordingly, theinvention provides a computer-based system for analyzing data producedusing the above methods in order to detect or predict a CIN lesion.

In certain embodiments, the methods are coded onto a physicalcomputer-readable medium in the form of “programming”, where the term“computer readable medium” as used herein refers to any storage ortransmission medium that participates in providing instructions and/ordata to a computer for execution and/or processing. Examples of storagemedia include floppy disks, magnetic tape, CD-ROM, a hard disk drive, aROM or integrated circuit, a magneto-optical disk, or a computerreadable card such as a PCMCIA card and the like, whether or not suchdevices are internal or external to the computer. A file containinginformation may be “stored” on computer readable medium, where “storing”means recording information such that it is accessible and retrievableat a later date by a computer. Of interest as media are non-transitorymedia, i.e., physical media in which the programming is associated with,such as recorded onto, a physical structure. Non-transitory media doesnot include electronic signals in transit via a wireless protocol.

With respect to computer readable media, “permanent memory” refers tomemory that is permanent. Permanent memory is not erased by terminationof the electrical supply to a computer or processor. Computerhard-drive, CD-ROM, floppy disk and DVD are all examples of permanentmemory. Random Access Memory (RAM) is an example of non-permanentmemory. A file in permanent memory may be editable and re-writable.

Kits

In yet another aspect, the present invention provides kits forpracticing the subject methods, e.g., as described above. The subjectkits may include labeled biomarker probes, e.g., as described above. Inaddition, the kits may include non-specific stains, as described above.In addition, the kit may include one or more additional compositionsthat are employed, including but not limited to: buffers, diluents,fixing reagents, permeabilizing reagents, etc., which may be employed ina given assay. Kits may further include sample obtainment devices, e.g.,cervical brooms, as described above. The above components may be presentin separate containers or one or more components may be combined into asingle container, e.g., a glass or plastic vial.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

A 1 ml sample aliquot of the liquid-based cervical cytology (LBC)specimen obtained was and centrifuged at 1000×g for 5 minutes at roomtemperature. The resultant cell pellet was washed twice in phosphatebuffer saline (PBS), pH 7.4 and the cells were fixed and permeabilizedfor 1 h at room temperature using IncellFP (Incelldx, Menlo Park,Calif.). The resultant fixed and permeabilized cells were washed twiceusing two different pre-hybridization buffers (PBS and 2×SSC) and ahybridization cocktail was prepared by mixing hybridization buffer(5×SSC, 30% formamide) with a fluorescence-labeled HPV E6, E7 mRNA probecocktail for all known high risk HPV types (the probe cocktail was fromthe those found in the probe cocktail of the HPV OncoTect™ E6, E7 mRNADetection Kit (incellDx, Menlo Park, Calif.). The hybridization reactionwas performed in a preheated 43±1° C. water bath for 30 minutes and wasfollowed by stringency washing of cells with two post-hybridizationbuffers (2×SSC, Triton X-100 and 0.1×SSC, Triton X-100) in order toremove the unbound probe. The cells were washed in 1 ml PBS containing2% fetal calf serum and re-suspended in 60 μl PBS, 0.05% Triton X-100,and 1 μg/ml DAPI. Prior to running on the instrument, 200 μl 10 μM EDTAin PBS was added. The samples were homogenized with a syringe and run onan ImageStream instrument (Amnis Inc, Seattle, Wash.). The instrumentwas set-up to distinguish intact single cells using Aspect Ratio versusArea dot plot, e.g., as shown in FIG. 1. This setting allowed: (1)identification of abnormal cells by N/C (nuclear to cytoplasmic) ratioanalysis (see e.g., FIG. 2); and (2) quantification of E6, E7 mRNA asdetermined by DAPI staining and green fluorescence of the E6, E7 mRNAhybridization signal (see FIGS. 3A and 3B).

Alternatively, cell cycle analysis as determined by a DNA-stainingreagent (e.g., DAPI or DRAQ5 staining of the cells) and greenfluorescence of the E6, E7 mRNA hybridization signal can be used todetect abnormal cells. For example, as can be seen in the histogram inFIG. 4, overexpression of E6/E7 mRNA (Y-axis) is found in cells havingincreased DNA content (X-axis; DNA stain is DRAQ5).

Combined use of morphometric measurements consistent with accepted slidebased criteria in Table 1 and E6, E7 mRNA overexpression on acell-by-cell basis all performed on cells while in suspension increasedthe sensitivity and specificity for detection of CIN 2+ to >95% and >90%respectively. This performance in a single assay greatly exceeds thetest performance of the PAP smear and HPV DNA combined for the detectionof CIN 2+ lesions.

This approach can be used substituting a p16 antibody for E6, E7 mRNAdetection, or substituting E6, E7 mRNA probes with probes directed atspecific microRNAs or chromosome alterations 3q-associated with cervicalcancer, or substituting E6, E7 directed antibodies for E6, E7 mRNAprobes.

As an additional alternative, cell cycle analysis as determined by aDNA-staining reagent coupled with morphometric measurements, e.g.,nuclear/cytoplasmic ratio of the cells, can be used to detect abnormalcells. For example, FIGS. 5A and 5B show histograms of N/C ratios(Y-axis) versus DNA content of normal cervical cells (FIG. 5A, toppanel), LSIL cervical cells (FIG. 5A, bottom panel), and HSIL cervicalcells (FIG. 5B, both panels). This alternative uses only 1 stainingreagent, i.e., the non-specific DNA-staining reagent.

Furthermore, as shown in Table 2 below, we have found that degree ofnucleation of squamous cells in the sample, as determined by using a DNAstaining reagent, corresponds with the severity of the cytologicabnormality. Specifically, an increase in the percent nucleation of thesquamous cells in the sample corresponds to an increased severity of thecytologic abnormality. In Table 2, normal squamous cells had a %nucleation of about 50%, LSIL squamous cells had a % nucleation of about80%, and HSIL squamous cells had a % nucleation above 90%. As such, apercent nucleation for squamous cells in a sample from a subject ofabout 80% or higher indicates that the subject has a cervicalintraepithelial neoplasia (CIN) lesion (e.g., LSIL or HSIL), where apercent nucleation of about 90% or higher in the sample indicates thatthe subject has a high grade squamous epithelial lesion (HSIL).

TABLE 2 Nucleation of the Squamous Cell Population as a Determinate ofAbnormal Pap Smear Diagnosis Sample % group Cytology nucleated 1 Normal47.33 2 LSIL 82.66 3 HSIL 94.38 4 HSIL 93.41 LSIL-low grade squamousepithelial lesion HSIL-high grade squamous epithelial lesion

Cervical cell samples may include cell types (and other debris) that arenot relevant to the screening assays described herein, and as such itwould be advantageous to exclude them from the analysis. FIG. 6 shows adot plot of combined side (orthogonal) light scatter (Y axis) andelectronic volume (X axis) of cells in a cervical cytology sample. Thisplot shows that different cells (and other components) in the sample canbe delineated using these morphometric parameters. The four differentgates identify debris, ectocervical cells, endocervical cells, andpolymorphonuclear leukocytes (PMNS). Gating can be employed to identifycells of interest to screen according to aspects of the presentinvention (e.g., endocervical and/or ectocervical cells).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1-70. (canceled)
 71. A method of detecting the presence of apre-cancerous cell in a biomarker labeled liquid cellular sample, themethod comprising: flow cytometrically assaying an aliquot of thesample, the assaying comprising: a) identifying an abnormal cell basedon morphometric data, cell cycle data or a combination thereof obtainedfrom cells of the sample; and b) detecting whether the abnormal cell isa pre-cancerous cell based on biomarker quantitation data flowcytometrically obtained from the abnormal cell.
 72. The method accordingto claim 71, wherein the abnormal cell is identified based onmorphometric data comprising nuclear to cytoplasmic ratio quantitation,nuclear area quantitation or a combination thereof.
 73. The methodaccording to claim 71, wherein the abnormal cell is identified based oncell cycle data comprising DNA content quantitation.
 74. The methodaccording to claim 71, wherein identifying the abnormal cell furthercomprises electronic volume quantitation.
 75. The method according toclaim 71, wherein the method further comprises fixing and permeabilizinga liquid cellular sample obtained from a subject to produce a fixed andpermeabilized liquid cellular sample used to generate the biomarkerlabeled liquid cellular sample.
 76. The method according to claim 75,wherein the method comprises contacting the fixed and permeabilizedliquid cellular sample with a biomarker probe to generate the biomarkerlabeled liquid cellular sample.
 77. The method according to claim 76,wherein the biomarker probe is a nucleic acid biomarker probe.
 78. Themethod according to claim 71, wherein the biomarker of the biomarkerlabeled liquid cellular sample is a nucleic acid biomarker.
 79. Themethod according to claim 78, wherein the nucleic acid biomarker is aDNA biomarker.
 80. The method according to claim 78, wherein the nucleicacid biomarker is a RNA biomarker.
 81. The method according to claim 71,wherein the method further comprises staining the biomarker labeledliquid cellular sample with a DNA specific stain.
 82. The methodaccording to claim 71, wherein the method comprises detecting whetherthe abnormal cell is a pre-cancerous cell based on biomarkerquantitation data of two or more biomarker probes.
 83. The methodaccording to claim 82, wherein the method comprises contacting the fixedand permeabilized liquid cellular sample with a hybridization cocktailof two or more biomarker probes.
 84. The method according to claim 83,wherein the two or more biomarker probes comprise differentfluorophores.
 85. The method according to claim 83, wherein the two ormore biomarker probes comprise the same fluorophore.
 86. The methodaccording to claim 71, wherein the method further comprises flowcytometrically assaying the aliquot of the sample to measure the degreeof nucleation of the cells of the aliquot or a subpopulation thereof.87. A system comprising: a flow channel; a light source configured todirect light to an assay region of the flow channel; a first detectorconfigured to receive light from the assay region of the flow channeland produce morphometric data, cell cycle data or a combination thereof;a second detector configured to receive light from the assay region ofthe flow channel and produce biomarker data; and a signal processingmodule configured to: a) receive the morphometric data, cell cycle dataor combination thereof from the first detector to detect when anabnormal cell is present in the flow channel based on the morphometricdata, cell cycle data or combination thereof; and b) receive thebiomarker data from the second detector and output a result of whetherthe detected abnormal cell is a pre-cancerous cell based on thebiomarker data.
 88. The system according to claim 87, wherein themorphometric data comprises nuclear to cytoplasmic ratio quantitation,nuclear area quantitation or a combination thereof.
 89. The systemaccording to claim 87, wherein the cell cycle data comprises DNA contentquantitation.
 90. The system according to claim 87, wherein the systemcomprises a third detector configured for electronic volumequantitation.
 91. The system according to claim 87, wherein the systemfurther comprises a biomarker labeled liquid cellular sample.
 92. Thesystem according to claim 87, wherein the signal processing module isfurther configured to output a result of the degree of nucleation of thecells of a biomarker labeled liquid cellular sample.
 93. A kitcomprising: a fluorescently labeled nucleic acid biomarker probe thatspecifically binds to a nucleic acid biomarker; and a DNA specificstain.
 94. The kit according to claim 93, wherein the kit furthercomprises a permeabilizing reagent.
 95. The kit according to claim 93,wherein the kit further comprises a fixation reagent.
 96. The kitaccording to claim 93, wherein the kit comprises a permeabilizingreagent and a fixation reagent present in a single container.
 97. Thekit according to claim 93, wherein the kit further comprises a secondfluorescently labeled nucleic acid biomarker probe that specificallybinds to a second nucleic acid biomarker.
 98. The kit according to claim97, wherein the kit comprises a hybridization cocktail comprising thefirst fluorescently labeled nucleic acid biomarker probe and the secondfluorescently labeled nucleic acid biomarker probe.
 99. The kitaccording to claim 93, wherein the kit further comprises a sampleobtainment device for obtaining a cellular sample from a subject.